The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and the pausing of RNApII at developmentally regulated genes in embryonic stem cells. This project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the current funding period of the parent grant, four specific aims are being pursued. The first is to understand the two known termination pathways (for polyA and non-polyA transcripts) and how RNApII chooses between them. A second aim studies the biogenesis of short unstable transcripts produced by divergent transcription at many RNApII promoters. The third aim uses NET-Seq to probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth aim exploits our discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit, asking whether the different CTD modifications and functions need to occur in cis on the same CTD or can be split between two CTDs. This supplement will add one new specific aim attempting to answer whether there are functional differences between CTD repeats at different positions. In the first subaim, a CTD will be engineered that adds several lysines and non-consensus repeats so that mass spectrometry can be used to distinguish proximal and distal repeats. The in vivo phosphorylations on these CTD fragments will be analyzed in wild-type cells and mutants of various CTD modifying enzymes. In the second subaim, a series of CTD mutants will be constructed that incorporate a non-native, photoreactive amino acid in vivo. In vivo crosslinking and analysis of associated proteins will show whether CTD-associated factors preferentially associate with proximal or distal repeats. Together, the results of these experiments will show whether the CTD phosphorylation patterns and associated proteins are distinct or identical in different regions of the CTD.